3-keto-4-aza-delta5-steroids and method of preparing same



Unite States Patent O fiice S-KETO-4-AZ'A-A -STEROWS AND lVIETHOD OFPREPARING SAME 6 Claims. (Cl. 260-287 This invention is directed to anovel method of .preparing nitrogen containing derivatives of steroidcompounds by a procedure which substitutes a nitrogen atom for an oxygenatom in one of the rings of a steroid derivative. The invention is alsodirected to novel u-keto-fl'- azo-y-unsaturated steroid compounds. Thenovel method is particularly directed to the preparation of these novelenol lactams, although other useful steroid derivatives can be preparedfrom the enol lactams. The invention is also directed to the enollactone of progesterone as a novel compound.

The new method of my invention involves heating in the presence of asource of ammonia or a primary amine a compound having acyclopentano-lO,l3-dirnethylpolyhydrophenanthrene structure except thatone of the nuclear rings contains an oxygen atom, and containing a ketogroup adjacent to the oxygen atom on one side, and a carbon-to-carbondouble bond adjacent to the oxygen atom on .the other side. The sourceof ammonia is preferably ammonia water, i.e., an aqueous solution ofammonia which is ammonium hydroxide.

My invention provides a simple, convenient method for replacing thehetero-oxygen atom of an nt-keto-B-oxa- 'y-unsaturated steroid with anitrogen atom. This procedure is very useful when it is desired toprepare a therapeutic aza steroid from an available oxa steroid. Even ifthe lactone of a given steroid is not available, many such lactones canbe readily prepared by known methods, and the present invention providesa method of inserting the nitrogen atom into the steroid ring, withoutthe use of any reducing agents so that the unsaturated bond adjacent tothe oxygen atom does not become saturated. Although the conversion ofsteroids to ot-keto- ,B-oxa-y-unsaturated steroids is old, it was notknown until the present invention that a-keto-fl-aza-y-unsaturatedsteroids could be prepared from steroids by an efiicient procedureinvolving conversion of the steroid to the keto acid, thence to theot-keto-,B-oxa-'y-unsaturated steroid, and finally to theu-ketto-fi-aza-qunsaturated steroid.

My steroids, particularly A -3-keto steroid derivatives in which anitrogen atom replaces the carbon atom in the position numbered C in thesteroid nucleus, are novel compounds of valuable therapeutic activity.

The a-keto-[i-oxa-v-unsaturated steroids which I employ are ordinarilyprepared from the keto acids of the steroids by heating in the presenceof acetic anhydride and acetyl chloride (R. B. Turner, J.A.C.S. 72, 579(1 950)) or by heating the keto acids in the presence of aceticanhydride and sodium acetate. The keto acids are prepared by ozonolysisof a steroid containing a conjugated carbonyl group, i.e.,

anti,

in vthe steroid nucleus. Some steroids containing conjugated carbonylgroups can also be oxidized to keto acids Patented July 28, 1959 byhypohalite solutions, e.g., A -cholesten-3-one is oxidized to the ketoacid by sodium hypobromite. I

The scrim of reactions to prepare the oc-keto-B-aza-qunsaturatedpolyhydro-IO,13-dimethylcyclopentanophenanthrenes can be represented bythe following reactions with progesterone:

out on, (i=0 =0 oxidation l dehydration .(IJHi III The steroid (1)containing conjugated carbonyl unsaturation is oxidized tocause ringcleavage and formation of the .keto acid (11), which is heated underdehydrating conditions to cause ring closure and formation of (III),wherein the lactone oxygen is in the position originally occupied by anunsaturated carbon atom in the steroid, and adjacent to the carbonylcarbon atom. The compound -(-III) is then heated in the presence ofammonia-to produce (IV), the nitrogen atom being in the positionoriginally occupied by the unsaturated carbon atom adjacent to thecarbonyl group.

The procedure of the present invention is .generally applicable tou-keto-B-oxa-y-unsaturated steroids. It will be understood of course,that the norand homosteroids are included in the group of steroids;e.g.,the 18- nor, l9-nor, A-nor, 'D-nor, "D-homo and D-' bis-l 1omosteroidscan be used. The process is also applicable to thecyclopentano-lO,l3 dimethylpolyhydrophenanthrenes regardless of the typeof side chain on the 17-carbon atom; for-example, the procedure can beapplied to the u-ketofl oxa-y-unsaturated derivatives of the sapogenins,the sitosterols, the stigmasterols, the ergosterols, the sexualhormones, etc. In some cases, it will be necessary to protect reactivegroups in the side chain by known methods during the ozonization step,but once the oxa cqmpound is prepared, the amination step can ordinarilybe conducted without protecting such groups. The ste-' roids can alsocontain other substituents, such as keto groups, hydroxyl groups,acyloxy groups, unsaturated groups, halo groups, etc. The step ofheating with ammonium hydroxide will not ordinarily affect such groups,except in a few cases; e.g., acid groups will react to give the amide orammonium salt derivatives; however, even in these cases ana-keto-B-aza-y-unsaturated steroid is still produced.

The procedure of the present invention can beapplied the reaction can beobtained if the-rings are expandedr for example, a D-homo steroid has aD-ring which is similar to the A-ring so far as obtaining thea-keto-fl-oxa- 'y-unsaturated structure is concerned.

EXAMPLE 1 infrared analysis showed a carbonyl group at 6.03 m

and an NH group at 3.23 my.

A -4-oxacholesten-3-one above was prepared by dissolving 8.4 grams of A-cholesten-3-one in a solution of 120 ml. of freshly distilled ethylacetate and 120 ml. of acetic acid. The solution was cooled to -10 C.and ozone was passed in at a setting of 100 volts, at a rate of 0.04cu.ft./min. and under 8 lbs. pressure, for 50 minutes, white crystallineprecipitate being formed after 30 minutes. Water, 40 ml., and 4 ml. of30% hydrogen peroxide were added, and the mixture was allowed to standovernight. The keto acid product was worked up by alkaline extraction,acidification of the extracts, extraction with ether, evaporation of theether, and recrystallization of the residue from ether-petroleum etherto give 5.79 grams of the keto acid of cholestenone of M.P. 144-147 C.The keto acid, 2 grams, was refluxed in 25 m1. of acetic anhydride and10 ml. of acetyl chloride for 64 hours. The solvent was evaporated underwater pump vacuum at 100 C. The residue was taken up in ether, washedwith water and bicarbonate, dried over Na SO and the ether solution wasevaporated to dryness. The residue was recrystallized from acetone-waterto yield 0.246 gram of the A -4-oxacholesten-3-one of M.P. 83 86 C.Another recrystallization from acetone-water solution raised the meltingpoint to 86-88 C.

EXAMPLE 2 Progesterone, 10 grams, was dissolved in a solution of 100 ml.of ethyl acetate and 100 ml. of acetic acid and ozonized at 100 volts,0.4 cu.ft./min., and 8 lbs. pressure at 10 C. for 45 minutes. Thereaction mixture was evaporated to dryness, and the procedure wasrepeated using a 30-minute ozonization period. Then 5 ml. of 30% H 0 in50 ml. of water was added and the mixture was allowed to stand for 18hours at room temperature after which a large quantity of ether wasadded. The ether solution was washed with water and then extracted withthree 100 ml. portions of dilute potassium hydroxide. The potassiumhydroxide extracts were combined and acidified with dilute hydrochloricacid. The precipitate was taken up in ether, washed with water, driedover Na SO and evaporated to dryness. The solid residue was dissolved in500 ml. of ether which was then concentrated to 75 ml. Precipitation wascaused by adding 25 ml. of hexane to yield 1.6 grams of the keto acid ofprogesterone of M.P. 168170 C.

The above procedure was repeated, starting with 12 grams ofprogesterone.

The keto acid of progesterone, 6 grams, was heated at 80 C. for 2 hoursunder nitrogen with 100 ml. of acetic anhydride and 40 ml. of acetylchloride. The reaction mixture was cooled and distilled under vacuum.The residue was dissolved in acetone, treated with charcoal (Darco)filtered, and the filtrate was concentrated. Petroleum ether (Skelly B)was added to precipitate the product which was either 4-oxa-A-pregnene-3,20-dione or a similar material with a partially dehydratedand acetylated side chain, i.e., having a o 7 -o(0 oH, =oH, group on the17-position.

The above product, 2 grams, was added to ml. of concentrated NH OH andthe mixture was heated for 18 hours in a bomb at 200 C. The mixture wascooled, evaporated to dryness, and recrystallized from an ethanolwatersolution to yield 0.1813 gram of M.P. 282-286 C. (with decomposition).The A -4-azapregnen-3,20-dione Was recrystallized to give a meltingpoint of 293300 C. (with decomposition).

EXAMPLE 3 A -4-oxapregnen-3,20-dione; 6.0 grams, and 90 ml. orconcentrated NH OH were placed in a bomb and heated at 200 C. for 42hours. The reaction mixture was cooled and filtered and the solidprecipitate was recrystallized from ethyl alcohol. Thefirst crop ofcrystals, M.P. 296- 302 C. (dec.), amounted to 1.685 grams. The secondcrop, of M.P. 292298 C. (dec.) and 0.788 gram in amount had thefollowing analysis:'

Calcd. for C H 9O NFC, 76.15; H, 9.26;N, 4.44. Found: C, 76.00; H, 9.40;N, 4.78.

The A -4-azapregnen-3,20-dione, exhibited marked antiinflaminatoryactivity in tests with rats with inflammed ankles. I

The table below compares the anti-inflammatory activity of the compoundin the Winters modification of the Selye inflammation test with ratswith inflamed ankles, to the activity of cortisone in that test. Thedosages were 50 mg./kg. subcutaneously.

Table I Ht" Compound 24 hours 48 hours AM-azapregnen-3,20-dione .J 7. 456. 65 Oortisone acetate 2. 59 2. 73

EXAMPLE 4 Cortisone acetate, 5.0 grams, was dissolved in 700 ml. of dryethyl acetate and cooledin a Dry Ice-methanol solution. Ozone was passedthrough the mixture for 45 minutes during which time the solution becamelight blue in color. The ozonolysis was stopped and 7.5 ml. of 30% H 0was added to the solution followed by 7.5 ml. of methanol. The solutionwas kept at room tempera,- ture for 72 hours. The material was worked upand crystallized to give 3,5-seco-17a-1ydroxy-2l-acetoxy-S,11,20-triketo-pregnane-3-oic acid of M.P. 158-159 C.

Analysis for C I-I O -L5H O: Calcd: C, 58.77; H, 7.39. Found: C, 58.88;H, 7.31.

The keto-acid product was suspended in 60 ml. of acetic anhydride and200. grams of sodium acetate was added. The solution was refluxed for 1hour, cooled, and evaporated to dryness. The residue was extractedsuccessively with several portions of ethyl ether and several portionsof ethyl acetate. The extractswere combined, washed, and dried. Thematerial was filtered and the filtrate was evaporated to dryness. Theresidue was dissolved in phenol and chromatographed on a column ofacidic A1 0 Methanol, ml, separated a cream colored solid, the4-oxa-17a-hydroxy-21-acetoxy A pregnene- 3,11,20-trione..'

EXAMPLE 5 The ke o acid of cortisone cet te s conver ed to the -xa-1)1-diacetexy-A -pregn ne-3, ,2 on or he 4-oxa=17a-hyd.roxy-21-acetoxy A-pregnene-3,1 1,20-t ione, depending. upon the reaction time, accordingto the pro eedlll'c of Solaway et al J.,A. .-S-v 7 5442 (1953). Both themonoand the diacetoxy compounds are converted to the 4.-aza derivativesaccording to. the proedu o Exampl 4 u ing reacti n. mes of t 0 hours.

The 4-aza deri tives of cortisone c n a so b p pared by heating the ketoacid of cortisone with ammonium hy roxid n a bomb for x o rs 150 C- asdescribed in my copending application, Serial No. 572,237, filed of evendate herewith.

EXAMPLE 6 Th pr edu e. of Ex mpl 1 was carri d out with7-ketocholesterol which can be obtained by oxidation ofcholesterolacetate with chromic acid (Windaus-et al., Ann. 520, 98(1935)). The ozonization procedure first converts the 7-ketocholesterolto 3:acetoxy-5,7-seco-.5.- keto-cholestaneJ-oic acid, which is thenconverted by ux ng in etic an ydr with i m. aceta e t he 4-oxaderivative and then by heating with ammonium hydroxide to 3-acetoxya6aza=A cholesten-7=one.- This compound can be readily saponificd t0 thecorresponding 3-hydroxy compound. 7

EXAMPLE '7 Testosterone'acetate was ozonized to the-corresponding ke'toacid by passing ozone into a solution of 7.29 grams of the acetate, in400 ml. of ethyl acetate and 100 ml. of acetic acid, at a rate of 0.4cu. ftJmin. under 8 lbs. pressure and at a setting of 100 volts, for- 45minutes at -60", C. The solution was warmed to C. and 400 ml. of; aceticacid and 5 ml. of hydrogen peroxide in 40 ml. of water was added, andthesolution. was allowed to stand for 18 hours. The reaction mixture wasworked up as in the previous examples, and the product wasrecrystallized from ether-acetone to give the keto acid of testosteroneacetate of melting point, 2Q2- 205 C. The keto acid is converted to the3.-keto-4-oxa- A .-de rivative of testosterone acetate by refluxing withacetic anhydride and sodium acetate and this is converted to the3-keto-4-aza-A -derivative of testosterone acetate by heating for 18hoursat 200 C. in the presence of 20 parts of ammonium hydroxide. Uponrecrystallization from ethyl alcohol, the compound has a melting point(with decomposition) of 306-308 C. By ordinary saponificationprocedures, the product is converted to the corresponding derivative oftestosterone, i.e., 4=aza- 17p-hydroxy-A -androsten-3-one.

EXAMPLE 8 The compound has the formula: t

dazal-A' Jl-D-homeeandrosfiatrien-3 one The ozonator used in the presentexamples was calibrated by running for 2 hours at 0.04 cu. ft./min.,volts, and 8 lbs. pressure, and collecting the ozone in 400 ml. of 2%KI, acidifying and titrating with 0.1062 N Na s- 0 154.8 ml. beingrequired.

EXAMPLE 9 Upon heating 1 gram of the A -4oxacholesten-3-one and 70 ml.of N-butyl amine at 200 C. for 18 hours, the N-butyl A-4-aza-cholesten-3-one is produced as a dark colored oil.

EXAMPLE 10 A -4-oxacholesten-3-one, 1' gram, was acetylated by refiuxingfor 2 /2 hours in 30 ml. of pyridine and 15 ml. of acetic anhydride. Thereaction mixture was evaporated to dryness and recrystallized frommethanol after treatment with charcoal, and had a M.P. of -117 C. Thecompound was recrystallized from methanol, M.P. l16l18 C. The N-acetyl A-4-azacholesten-3-one had the following analysis:

Calcd. for C H NO C, 78.63; H, 10.60; N, 3.27. Found: C, 78.62; H,10.49; N, 3.45.

Ultraviolet analysis gave max of less than 220 m and infrared gave noabsorption, acetate group absorption at 5.85 m and 7.97 m and lactamcarbonyl absorption at 5.95 m

EXAMPLE ll A 4-azapregnen-3,20-dione was acetylated by heating 0.3 gramof the compound to reflux for two hours in a solution of 15 ml. pyridineand 5 ml. of acetic anhydride. The N-acetyl product was recrystallizedfrom methyl alcohol.

The 3-keto-4-aza-A compounds of he present invention can be converted to3.keto-4-aza-derivatives by red c ion e P l d m cataly or by B uveal -Blreduction, the keto group can be reduced to a hydroxy g o p or a hy eneoupa A few of the steroids, other than those set forth the examplesabove, which can be used in the present in vention are:2-oxa-A31cholesten-l-one, 2-oxa-A -pregnadien-1,1l,20-trione,2-oxa-175-hydroxy-A -testen-l-one, 3- 0xa-A -pregnen-4,2O-dione, themethyl ester of 4-oxa-3 keto-A -testene-l7 8-carboxylic acid, 6-oxa-A-pregnen7, 20-dione, fi-oxa-N-pregnen-Zll-dione, 11-oxa-4,5-dibromo-l7a,2l-diacetoxy-A -pregnen-3,12,20-trione, l6- oxa-A-testen-l7-one, 4-oxa-A -ergostadiene-3-one, 4-oxa-11-acetoxy-A-pregnadien-3,20-dione, l9-nor-10e- A .-4-oxapregnen-3,20-dione, etc.

The above 3.-keto-4-aza-A compounds react respectively to give thedesired, novel 3-keto-4-aza-A compounds, i.e., 2-aza-A -chloesten-l-one,2-aza- A -pregna dien-l,1l ,20-trione, 2-aza-17B-hydroxy-A-testen-3-one, 3- aza-A -pregnen-4,20-dione, the methyl ester of4-aza-3- keto-A -testen-17,6-carboxylic acid, 6-aza-A -pregnen-7,20-dione, 6-aza-A -pregnen-7,ll-dione, ll-aza-4,5dibromol7a,21-diacetoxy-A-pregnen-3,12,20-trione, 16-aza-A testen-l7-one, 4-aza-A-ergostatrien-3-one, 4-azall acetoxy-A -pregnadien-3,20-dione,l9-nor-10e-A -4- azapregnen 3,20-dione, etc. The N-alkyl, N-aryl,N-acyl,

known methods Preferred N-acyl derivatives are those resulting fromreaction with formic, acetic, propionic,

butyrie, valer-ic, isovaleric, hexanoic, heptanoic, octanoic,

cyclohexanoic, benzoic, toluic, salicylic and naphthoic acids, acidchlorides, and acid anhydrides, especially those acyl derivatives inwhich the acyl group has 1 to 8 carbon atoms.

Although the present process can be applied to substitute nitrogen atomsin any of the rings of the steroid, it is more readily applicable tosubstitution in the A-ring, because of the larger number of naturalsteriods which possess structures in the A-ring which can be cleaved toproduce the keto-acids, and thereafter reacted to produce the3-keto-4-oxa-A compounds. Some of the preferred El-keto-4-oxa-Acompounds for use in my process can be represented by the followingstructural formula:

in which X is selected from I. .RI

in which R is -H, OH,

oiia and R" is COOR,

gl/ N z in which X has the same meaning as above and in which R ishydrogen, and alkyl, aryl, aralkyl, or acyl group.

The novel compounds of my invention have valuable uses for therapeuticpurposes. For example, some of my new compounds exhibitanti-inflammatory activity in tests which are related to activityagainst rheumatoid arthritis; for example A -4-azapregnen-3,2O-dioneexhibits marked activity in these tests, and A-17a-acetoxy-4-azatesten-3-one approaches the activity of cortisone. Mynew compounds have further uses in the preparation of other valuableorganic compounds for therapeutic and other purposes.

My novel compounds can be prepared as disclosed and claimed in thepresent application. Or, the new compounds can also be prepared fromketo acids or keto acid esters by the method disclosed and claimed in my00- pending application, Serial No. 572,237 filed of even date herewith.For example, the 3-keto-4-aza-A derivative of testosterone acetate canbe prepared by heating the keto acid of testosterone acetate with 20volumes of ammonium hydroxide at 200 C. for 18 hours. The N-butyl3-keto-4-aza-A derivative of cholestenone can be prepared by'heating 1gram of the keto acid of cholestenone and 70 ml. of N-butylamine to 200C. for 18 hours, and 4-aza-3-keto-D-homo-A -androstatriene can beprepared by heating 1 gram of 3,5-seco-5-keto-D-homo- A-androstadien-3-oic acid with 60 ml. of concentrated ammonium hydroxidefor 18 hours.

Primary amines, such as lower alkyl or aralkyl amines can be used inplace of ammonia to prepare N-alkyl or N-aralkyl lactams. For example,the following primary amines can be used; methyl amine, ethyl amine,n-propyl amine, n butyl amine, isopropyl amine, 2-methyl-3-amino-propane, n-pentyl amine, n-hexyl amine, Z-methyl- 4-amino-butane,n-octyl amine, cyclohexylamine, cyclohexylmethylarnine, ethanol amine,l-aminohexene-S, (2- phenylethyl) amine, (l-phenylethyl) amine, etc. Thearomatic primary amines, e.g., aniline, p-ethylaniline, naphthylamines,etc., are less preferred because they are more. ditficult to react, butthey can be used in the reaction.

While ammonium hydroxide is ordinarily the source of ammonia, it shouldbe understood that ammonia gas can simply be passed over or through thesteroid at the reaction temperature. While it is convenient to conductthe reaction under autogenous pressure in order to avoid the loss ofvolatile reactants, this is not'considered essential to the process asthe ammonia gas can be recycled and kept in contact with the steroid atlower pressures.

The reaction conditions for the formation of the 3- keto-4-aza-Acompound can vary considerably; for example, temperatures of -250" C.can be used for 6 to 60 hours. Temperatures of to 210 C. for 15 to 50hours are ordinarily used. While a commercial concentrated ammoniumhydroxide is ordinarily used for convenience, it is possible to usedilute ammonium hydroxide solutions, or more concentrated ammoniumhydroxide, or ammonia itself. The primary amines can be used as such ordissolved in solvents. Some water should be present if it is desired toavoid the conversion of keto groups to imide groups; the water liberatedin the reaction is usually suflicient for this purpose.

A method of preparing 3-keto-4-azo-A derivatives of steroids fromsteroids having conjugated carbonyl unsaturation, via the 3-keto-4-oxa-Aderivatives of steroids has been described. A new class of valuablechemical compounds, the 3-keto-4-aza-A derivatives of steroids has beendescribed.

I claim:

1. As a new compound, A -4-azapregnen-3,ZO-dione.

2. As a new compound, A -17a-acetoxy-4-aza-testen- 3-one.

3. A method of preparing 3- keto-4-aza-A -steroids which comprisesheating a compound of the structure:

in which X is selected from the group consisting ofv and and R isselected from the group consisting of H, OH and and R" is selected fromthe group consisting of O OH|(OH)p2- H, OH, CH CH CH(CH (CH CH(CH RCOOand --CH(CH )CH=CHCH(CH )--CH--(CH and R is selected fi'om the groupconsisting of H and lower alkyl, to temperatures of 100 to 250 C. in thepresence of a compound selected from the group consisting of ammonia,methyl amine, n-propyl amine, n-butyl amine, isopropyl amine,2-methyl-3-amino-propane, n-pentyl amine, n-hexyl amine,2-methyl-4-amino-butane, n-octyl amine, cyclohexyl amine,cyclohexylmethyl amine, (2-phenylethyl) amine and (l-phenylethyl) amineto produce a compound of the structure:

iiiiiiiX in which X has the meaning set forth above, and R is,

References Cited in the file of this patent UNITED STATES PATENTS BoltJan. 7, 1941 Mazur Mar. 13, 1956 Knowles Aug. 28, 1956 OTHER REFERENCESKaufmann: J. Am. Chem. Soc., vol. 73, April 1951, pp. 1779-1780.

Turner: J.A.C.S., vol. 72, 1950, pp. 579-585.

Wagner et 211.: Synthetic Org. Chem. (N.Y.), John Wiley, 1953, page 576.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.2,897,202 July 28, 1959 Bernard S. Wildi It is hereby certified thaterror appears in the above numbered patent requiring correction and thatthe said Letters Patent should read as corrected below.

Column 2, lines 27 to 29, between formulas IV and III,

for

NH OH read NH OH heat heat Signed and sealed this 9th day of April 1963.

(SEAL) Attest:

ESTON G. JOHNSON DAVID L. LADD Attesting Officer l Commissioner ofPatents

1. AS A NEW COMPOUND *5-4AZAPREGNEN-3,20-DIONE.
 2. AS A NEW COMPOUND,*5-17 A-ACETOXY-4-AZA-TESTEN3-ONE.
 3. A METHOD OF PREPARING3-KETO-4-AZA-*5-STEROIDS WHICH COMPRISES HEATING A COMPOUND OF THESTRUCTURE: